A short workflow enables RNA purification with genomic DNA removal in less than 30 minutes.

High, reproducible RNA yields.

Total RNA was purified from 10-fold serial dilutions of Jurkat cells (5 x 105 to 5 cells) using the RNeasy Plus Micro Kit; at each dilution, RNA was purified from 4 replicates. β-actin transcript was detected in down to 5 cells by real-time RT-PCR, and reproducible CT values were observed at each dilution. In control reactions without reverse transcriptase (-RT), β-actin DNA was not detected after 38 cycles, indicating the absence of genomic DNA contamination.

Cells and easy-to-lyse tissues are first lysed and homogenized in highly denaturing guanidine-isothiocyanate–containing Buffer RLT Plus, which immediately inactivates RNases to ensure isolation of intact RNA. The lysate is then passed through a gDNA Eliminator spin column that, in combination with the high-salt buffer, selectively and efficiently removes genomic DNA. Ethanol is added to provide appropriate binding conditions for RNA, and the sample is applied to an RNeasy MinElute spin column. These specialized columns each contain a silica membrane that specifically binds RNA from lysed cells.

Procedure

Total RNA is purified from up to 5 x 105 cells or 5 mg tissue, making the RNeasy Plus Micro Kit well suited for small samples. A short workflow enables RNA purification with genomic DNA removal in less than 30 minutes (see flowchart "RNeasy Plus procedure"). Samples are first lysed and homogenized. The lysate is passed through a gDNA Eliminator spin column, ethanol is added to the flow-through, and the sample is applied to an RNeasy MinElute spin column. RNA binds to the membrane and contaminants are washed away. High quality RNA is eluted in as little as 14 µl water.

Different protocols are available for different starting materials. The protocols differ mainly in the lysis and homogenization of the sample. Once the sample is applied to the gDNA Eliminator spin column, the protocols are similar. The procedure provides an enrichment for mRNA since most RNAs <200 nucleotides (such as rRNAs and tRNAs) are excluded. The RNeasy Plus procedure can be modified to allow the purification of total RNA containing small RNAs, such as miRNA, from cultured cells.

When disrupting and homogenizing tissues in Buffer RLT Plus (supplied with the RNeasy Plus Micro Kit), excessive foaming may occur. This foaming is substantially reduced by adding Reagent DX (supplied separately) to Buffer RLT Plus before starting disruption and homogenization. Reagent DX has been carefully tested with the kit, and has no effect on RNA purity or on downstream applications.

Applications

RNA purified using the RNeasy Plus Micro Kit is ideally suited for downstream applications that are sensitive to low amounts of DNA contamination, such as quantitative real-time RT-PCR. The purified RNA can also be used in other applications.